Lf qc0103

We blocked blotted membranes with blocking buffer (5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% tween-20 (TBST)) for 1 hat room temperature, then incubated with primary antibodies (goat-anti E-cadherin, AF-748, R&D Systems, Minneapolis, MN, USA, 1:2000, mouse-anti AQP5, sc-514022, Santa Cruz Biotechnology, Delaware Avenue Santa Cruz, CA, USA, 1:2000, mouse-anti β-actin, A2228, Sigma-Aldrich, 1:2000) for 16–21 hat 4 C. After washing with TBST 10 min three times, and we incubated the membranes with HRP-conjugated secondary antibodies (goat-anti mouse, 115-035-141, 1:1000; donkey-antigoat, 705-035-147, 1:1000, Jackson ImmunoResearch, West Grove, PA, USA) for 1.5 hat room temperature. The immunoblotting was visualized using the enhanced chemiluminescence reagent (ECL; AbFrontier, LF-QC0103, Seoul, South Korea). When we used the membranes more than once, we removed antibodies with a low-pH stripping buffer (pH 2.2; 200 mM glycine, 1% SDS, 0.03% Tween-20) for 5–10 minat RT and then re-start the blocking step.

At days 0, 1, 3 and 7, animals were euthanized with a urethane intraperitoneal injection (1 g/kg), and both temporal bones were immediately removed. Two cochleae were homogenized in a tube containing ice-cold lysis buffer (100 mM Tris, pH 7.4, 200 mM NaCl, 1% NP-40, 10 mM MgCl2) with protease inhibitors. Western blotting was performed, as described previously.^{17 (link)} In brief, lysates were centrifuged for 20 min at 13 000 r.p.m. at 4 °C; in addition, proteins in the supernatant were separated using 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were incubated overnight with the primary antibodies against 4-HNE (1 : 1000 dilution), BDNF (ab6201, 1 : 1000 dilution; Abcam) and NT-3 (ab65804, 1 : 1000 dilution; Abcam). After rinsing in TBST, the membranes were incubated with the HRP-conjugated secondary antibody (81–1620, A16104, G21040; 1 : 5000; Invitrogen) for 1 h. Bands were detected by chemiluminescence (LF-QC0103, Ab Frontier, Seoul, Korea). Further, blots were stripped and reprobed with actin (SC-1616, Santa Cruz Biotechnology), thereby serving as a protein loading control. Band intensities were quantified using the ImageJ software (NIH).